Segmentation is required to quantify cellular structures in microscopic images. This typically requires their fluorescent labeling. Convolutional neural networks (CNNs) can detect these structures also in only transmitted light images. This eliminates the need for transgenic or dye fluorescent labeling, frees up imaging channels, reduces phototoxicity and speeds up imaging. However, this approach currently requires optimized experimental conditions and computational specialists. Here, we introduce "aiSEGcell" a user-friendly CNN-based software to segment nuclei and cells in bright field images. We extensively evaluated it for nucleus segmentation in different primary cell types in 2D cultures from different imaging modalities in hand-curated published and novel imaging data sets. We provide this curated ground-truth data with 1.1 million nuclei in 20,000 images. aiSEGcell accurately segments nuclei from even challenging bright field images, very similar to manual segmentation. It retains biologically relevant information, e.g. for demanding quantification of noisy biosensors reporting signaling pathway activity dynamics. aiSEGcell is readily adaptable to new use cases with only 32 images required for retraining. aiSEGcell is accessible through both a command line, and a napari graphical user interface. It is agnostic to computational environments and does not require user expert coding experience.

MOTIVATION: Recent imaging technologies allow for high-throughput tracking of cells as they migrate, divide, express fluorescent markers and change their morphology. The interpretation of these data requires unbiased, efficient statistical methods that model the dynamics of cell phenotypes. RESULTS: We introduce treeHFM, a probabilistic model which generalizes the theory of hidden Markov models to tree structured data. While accounting for the entire genealogy of a cell, treeHFM categorizes cells according to their primary phenotypic features. It models all relevant events in a cell's life, including cell division, and thereby enables the analysis of event order and cell fate heterogeneity. Simulations show higher accuracy in predicting correct state labels when modeling the more complex, tree-shaped dependency of samples over standard HMM modeling. Applying treeHFM to time lapse images of hematopoietic progenitor cell differentiation, we demonstrate that progenitor cells undergo a well-ordered sequence of differentiation events. AVAILABILITY AND IMPLEMENTATION: The treeHFM is implemented in C++. We provide wrapper functions for the programming languages R (CRAN package, https://CRAN.R-project.org/package=treeHFM) and Matlab (available at Mathworks Central, http://se.mathworks.com/matlabcentral/fileexchange/57575-treehfml). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Quantitative large-scale cell microscopy is widely used in biological and medical research. Such experiments produce huge amounts of image data and thus require automated analysis. However, automated detection of cell outlines (cell segmentation) is typically challenging due to, e.g. high cell densities, cell-to-cell variability and low signal-to-noise ratios. RESULTS: Here, we evaluate accuracy and speed of various state-of-the-art approaches for cell segmentation in light microscopy images using challenging real and synthetic image data. The results vary between datasets and show that the tested tools are either not robust enough or computationally expensive, thus limiting their application to large-scale experiments. We therefore developed fastER, a trainable tool that is orders of magnitude faster while producing state-of-the-art segmentation quality. It supports various cell types and image acquisition modalities, but is easy-to-use even for non-experts: it has no parameters and can be adapted to specific image sets by interactively labelling cells for training. As a proof of concept, we segment and count cells in over 200 000 brightfield images (1388 × 1040 pixels each) from a six day time-lapse microscopy experiment; identification of over 46 000 000 single cells requires only about two and a half hours on a desktop computer. AVAILABILITY AND IMPLEMENTATION: C ++ code, binaries and data at https://www.bsse.ethz.ch/csd/software/faster.html . CONTACT: [email protected] or [email protected]. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Cell fate decisions have a strong stochastic component. The identification of the underlying mechanisms therefore requires a rigorous statistical analysis of large ensembles of single cells that were tracked and phenotyped over time. RESULTS: We introduce a probabilistic framework for testing elementary hypotheses on dynamic cell behavior using time-lapse cell-imaging data. Factor graphs, probabilistic graphical models, are used to properly account for cell lineage and cell phenotype information. Our model is applied to time-lapse movies of murine granulocyte-macrophage progenitor (GMP) cells. It decides between competing hypotheses on the mechanisms of their differentiation. Our results theoretically substantiate previous experimental observations that lineage instruction, not selection is the cause for the differentiation of GMP cells into mature monocytes or neutrophil granulocytes. AVAILABILITY AND IMPLEMENTATION: The Matlab source code is available at http://treschgroup.de/Genealogies.html.

BACKGROUND: In recent years, high-throughput microscopy has emerged as a powerful tool to analyze cellular dynamics in an unprecedentedly high resolved manner. The amount of data that is generated, for example in long-term time-lapse microscopy experiments, requires automated methods for processing and analysis. Available software frameworks are well suited for high-throughput processing of fluorescence images, but they often do not perform well on bright field image data that varies considerably between laboratories, setups, and even single experiments. RESULTS: In this contribution, we present a fully automated image processing pipeline that is able to robustly segment and analyze cells with ellipsoid morphology from bright field microscopy in a high-throughput, yet time efficient manner. The pipeline comprises two steps: (i) Image acquisition is adjusted to obtain optimal bright field image quality for automatic processing. (ii) A concatenation of fast performing image processing algorithms robustly identifies single cells in each image. We applied the method to a time-lapse movie consisting of ∼315,000 images of differentiating hematopoietic stem cells over 6 days. We evaluated the accuracy of our method by comparing the number of identified cells with manual counts. Our method is able to segment images with varying cell density and different cell types without parameter adjustment and clearly outperforms a standard approach. By computing population doubling times, we were able to identify three growth phases in the stem cell population throughout the whole movie, and validated our result with cell cycle times from single cell tracking. CONCLUSIONS: Our method allows fully automated processing and analysis of high-throughput bright field microscopy data. The robustness of cell detection and fast computation time will support the analysis of high-content screening experiments, on-line analysis of time-lapse experiments as well as development of methods to automatically track single-cell genealogies.